EP1807696B1 - Dispositif et procede pour mesurer les proprietes de cellules - Google Patents

Dispositif et procede pour mesurer les proprietes de cellules Download PDF

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EP1807696B1
EP1807696B1 EP05801676A EP05801676A EP1807696B1 EP 1807696 B1 EP1807696 B1 EP 1807696B1 EP 05801676 A EP05801676 A EP 05801676A EP 05801676 A EP05801676 A EP 05801676A EP 1807696 B1 EP1807696 B1 EP 1807696B1
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cavity
measuring
opening
double
cell
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EP1807696A1 (fr
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Richard Wagner
Karsten Gall
Andreas Wirth
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Universitat Osnabrueck
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Universitat Osnabrueck
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells

Definitions

  • the invention relates to methods for detecting properties of cells and vesicles, and to measuring devices for use in these methods.
  • the patch-clamp technique is common.
  • a glass pipette (diameter about 1 micron) is filled with an electrolyte solution and carefully placed on the surface of a cell. After perforation of the membrane spots in the patch pipette, it is then possible to measure currents or the potential over the entire cell surface (whole-cell recording). But you can also, and this is the real advantage of the patch clamp technique, measure currents through individual channels that are located in the patch directly below the pipette tip.
  • a cell is aspirated onto a smaller opening between two superimposed chambers, with the aperture adapted to the cell size.
  • a reproducibly high sealing resistance can be achieved only in a single experiment and there only on a smaller number of membrane types, which greatly limits the applicability of the patch-clamp technique.
  • Modern drug discovery means that, despite many attempts at automation, it is still necessary to resort to hitherto non-standardized, primarily fluorescence-based methods.
  • fluorescence-based methods For the approval of drugs, in which pharmacological safety is given the highest priority, and which can be tested almost exclusively with electrophysiological measurement techniques, sequential single measurements represent a considerable hurdle. Screening of potential active substances on a large scale is not possible with this method , Optical measurements can not be performed on these automated patch clamp systems either.
  • Another established technique for acquiring electrophysiological data is vertical lipid bilayers stretched between two electrolyte-filled chambers (Borisenko et al., 2003, Hinnah et al., 2002). After fusion of ion channels or transporter proteins into the lipid bilayers, the protein mediated current can be resolved down to the single molecule level.
  • the established classical lipid bilayer technique is not automatable. The measurement of properties of membranes of intact cells is not possible in contrast to the patch-clamp technique. The technique is therefore not suitable and approved for the pharmacological testing of active substances, since only measurement methods are used on whole cells.
  • the US 2003/0146091 A1 describes a device for performing measurements on cells with one or more samples.
  • a hydrophilic partition wall for example of silicon nitride or silica.
  • the hydrophilic partition is partially coated with a hydrophobic coating so that the cell can be positioned so that its membrane joins the hydrophobic coating.
  • the multilayer device is relatively complicated to produce. The positioning of the cells on the device is susceptible to interference, since the measurement objects themselves, ie the cells, must first seal all openings of the device in order to achieve a "gigaseal" and thus be able to make measurements.
  • lipid bilayers containing fusion factors with cells expressing cell fusion proteins on the surface.
  • a measuring chamber is used with a hydrophobic partition wall, which contains an opening of 150-200 microns.
  • a lipid bilayer is incorporated which contains essential factors such as cholesterol and sphingolipids for fusion.
  • the establishment of this system is complicated because the special fusion factors have to be added or expressed.
  • the DE 10047390 A1 describes devices with a plurality of measuring chambers, each shared by a lipid bilayer. After application of a voltage through the lipid bilayers, changes in the membranes upon addition of organic compounds can be investigated.
  • the device is suitable for high throughput screening.
  • the device has disadvantages: Thus, the disassembly of the device and the application of the bilayer according to Fig. 1b is cumbersome.
  • the lower sample chambers are not easily accessible and difficult to fill and clean evenly.
  • the silver electrodes are arranged so that optical measurements are not possible. Since the lower chambers are completely closed after the formation of the lipid bilayer, when samples are added to the chamber above the membrane an osmotic pressure can be produced which impairs the membranes and thereby falsifies the measurements.
  • the object of this invention is the development of a novel micro- or nanoanalytical technology that overcomes the disadvantages of the above-described methods.
  • a method and auxiliaries are to be provided in order to detect cell properties such as electrophysiological and optical properties accurately, reliably and in the simplest possible and reproducible manner.
  • the method should be automatable and already be used in the early primary screen of drugs.
  • the US-A-2002/0064841 discloses ion electrodes, in particular microelectrodes and electrode arrays, and methods for producing such electrodes.
  • Planar polymer electrodes are used for patch-clamp measurements of ion currents through biological membranes, for example plasma membranes, of living cells.
  • the electrodes described there are useful for measuring individual and multi-sided cell membrane currents and voltages and can also be used in "high-throughput screening" methods.
  • lipid bilayer membranes have been used as a model system for the study of lymphocyte-mediated target cell elimination.
  • Lipid bilayers that are dinitrophenylated can stably support dozens of lymphocytes without breaking or increasing the electrical conductivity of the membrane.
  • human lymphocytes rapidly caused an increase in membrane conductivity in several orders of magnitude without breaking the membrane.
  • electrophysiological experiments and no observations of changes in the cell membrane are disclosed by optical methods. With the measuring chambers disclosed in this work, this is also not possible in principle.
  • the invention discloses a measuring device for detecting at least one property of cells or vesicles divided from a hydrophobic partition having a horizontal opening (measuring aperture) into two chambers containing electrolyte solution, the aperture being closed by a lipid bilayer , At least one cell or vesicle contacts the lipid bilayer.
  • a lipid bilayer (lipid bilayer, bilayer) is stretched over the opening.
  • This lipid bilayer forms spontaneously when biological, purified naturally occurring or synthetic lipid is suitably applied to a hydrophobic substrate.
  • the lipid bilayer seals the opening and thus both chambers against each other with a resistance of typically 10 10 ⁇ .
  • the stability of the lipid bilayer over time is inversely proportional to the radius of the opening and is already in the range of hours to days for holes with a diameter of 30 ⁇ m (Hinnah et al., 2002).
  • the contacting takes place in that the lipid bilayer is arranged horizontally.
  • the cell or vesicle is then placed on or over the lipid bilayer, it is primarily in contact with the lipid bilayer by gravity.
  • the contacting may also be otherwise effected or enhanced, for example by movement of the measuring device, the electrolyte solution and / or by utilizing attractive interactions between the lipid bilayer and the cell or the vesicle.
  • the contacting can also be effected or enhanced by the use of suitable receptor / ligand systems or surface proteins that promote cell fusion. However, such targeted interactions are not required for the disclosed system.
  • the cell / membrane fusion is not artificially enhanced by the addition of appropriate fusion components or by prior targeted expression of protein components that cause or promote the fusion, such as the E1 and E2 envelope proteins.
  • appropriate fusion components such as the E1 and E2 envelope proteins.
  • protein components that cause or promote the fusion such as the E1 and E2 envelope proteins.
  • the addition of the vesicles to a lipid bilayer alone creates conditions which permit the measurement of the vesicle properties in a simple manner.
  • the molecular mechanisms of the coupling are not known in detail, but their result is a coupling of the cell with the lipid membrane, in which the cell largely its morphological structure maintained and together with the bilayer represents a common electrical resistance (ohmic coupling).
  • the lipid membrane of the cell or vesicle partially or completely fuses with it after contacting the lipid bilayer.
  • the detection of cell characteristics preferably occurs when the fusion is complete and the system has stabilized.
  • the disclosed method is applicable even when a cell or vesicle is in contact with a lipid bilayer, and both form a common capacitance (capacitive coupling) without fusion of the membranes. Already such an arrangement allows z.
  • Example the measurement of electrical properties of the cell, which acts like a capacitor when applying a voltage across the lipid bilayer.
  • the horizontal opening has a diameter of 0.1 to 100 .mu.m, in particular 0.5 to 50 or 2 to 40 microns.
  • the opening is preferably round or oval.
  • the lipid bilayer positioned in the opening is substantially horizontal due to the opening. This means that it can be slightly curved up or down. Such a curvature can arise, for example, if the solutions above and below the membrane are different.
  • the hydrophobic partition may or may not be horizontal. It may, for example, have a slight inclination towards the measuring opening so that a cell which is introduced into the first cavity (11) passes by gravity in the direction of the lipid bilayer.
  • the slope of the hydrophobic partition to the horizontal is less than 45, 20 or 10%.
  • the hydrophobic partition is preferably a polymer film.
  • the partition is made of teflon, PMMA, PDMS, topaz or polycarbonate.
  • the partition preferably has a thickness of 1-100 microns.
  • materials that are sufficiently non-polar are suitable so that a lipid bilayer can be stably introduced in an opening in the material.
  • "Hydrophobic" in connection with the dividing wall means that a stable lipid bilayer can be formed in an opening in the dividing wall.
  • the electrolytic solution is preferably a physiological saline solution.
  • salts in particular those are suitable that the ions K +, Na +, Mg 2+, Ca 2+, Cl - contain, SO 4 2- and PO 4 3-.
  • the pH is preferably between 5 and 9 and is adjusted with suitable pH buffer substances.
  • the lipid bilayer consists of synthetic and / or purified biological lipids.
  • the generation of lipid bilayers in openings in partitions for performing electrophysiological measurements is known in the art, for example, Hinnah et al., 2002, incorporated herein by reference.
  • a lipid preparation is used to produce the lipid bilayer from synthetic or purified biological lipids.
  • the lipids are no longer in their natural environment, ie no longer constituents of the intact cell organelle, cell or other naturally occurring vesicle.
  • a circuit is applied through the measuring device and through the lipid bilayer, so that measurements can be made regarding electrical properties of the lipid bilayer and the cells in contact with the lipid bilayer.
  • the measuring device consists at least partially of optically transparent material, so that optical measurements can be carried out. For example, it can be examined whether the cell emits an optical signal upon addition of a potential receptor ligand.
  • the inventive method is particularly suitable for automated implementation.
  • the invention also provides an array for the automated detection of properties of cells or vesicles, comprising at least two measuring devices.
  • the measuring devices are preferably firmly connected to each other and individually addressable electrically.
  • the arrays according to the invention allow the implementation of the method at least partially by robots or machines. They comprise at least 2, preferably 50 to 2000, especially 96, 384, or 1536 measuring devices (SBS standard).
  • the invention allows the utilization of microscopic horizontal lipid bilayers as support for whole cells. These investigational objects, which are important in drug discovery, can be analyzed not only with the fluorescence-based methods already established in high-throughput screening (HTS), but simultaneously also electrically.
  • HTS high-throughput screening
  • the individual support lipid bilayers can be miniaturized to a high degree and measured at the same time or in a timely manner in nanotechnologically structured and fabricated arrays (nanoarrays).
  • nanotechnologically structured and fabricated arrays nanoarrays
  • the new technology will be the analysis significantly expand membrane transport processes.
  • this invention does not use patch pipettes which require a stable sealing resistance.
  • the sealing resistance forms spontaneously and stably at least for hours.
  • this connection between lipid bilayer and hydrophobic film has the high mechanical stability necessary for HTS.
  • the positioning of the cells on the Lipiddoppelschift is preferably carried out simply by adding this above the lipid bilayer.
  • the method described here is also much cheaper than that described in the prior art, since the measuring chambers can be made parallel in sandwich construction and contain no mechanically unstable components such as vacuum lines.
  • optical path to the cell is fully usable, so that this invention can be combined with high-resolution fluorescence microscopy and spectroscopy.
  • the coupling between the lipid bilayer and cells makes it possible, with a sealing resistance of, for example, 10 8 - 10 10 ⁇ , to measure the electrical properties of the membranes.
  • the method also allows parallel optical measurements. Accurate positioning of the cell on the lipid bilayer is not necessary, making the method highly automated and parallelizable.
  • the invention also provides double and triple measuring chambers and microtiter plates and methods according to one of claims 3 to 8 and 10.
  • the double and triple measuring chambers according to the invention are particularly suitable for use in a method according to the invention for determining cell properties. However, they can also be used generally to study properties of lipid bilayers without contacting cells.
  • FIG. 5a shows a einclosystemzu erfindungmä on method with a first cavity (11) with lateral walls (4) and a partition wall (3), which is also the bottom of the cavity. Below a second cavity (12) is arranged, which is connected to the first cavity (11) through a measuring opening (13). The second cavity (12) is bounded laterally by the layer (3) and by a lower bottom (1). Metal electrodes are positioned in both cavities.
  • the bottom floor is made of an optically active material such as glass.
  • FIG. 5b shows a dual-chamber system according to the invention with two first cavities (11) and (15) and a continuous second cavity (12), a first measuring opening (13) and a second opening (14).
  • FIG. 5c shows a three-chamber system according to the invention with the features according to FIG. 5b ,
  • a further first cavity (16) is included with an opening (17).
  • the second cavity (12) extends continuously below the first cavities (11), (15) and (16).
  • FIGS. 5a and 5c show the double or triple chamber system according to the invention with the features according to the invention FIGS. 5b and 5c .
  • two electrodes (21) and (22) are included, which are connected via a voltage source U (23) and an ammeter I (24).
  • U voltage source
  • I ammeter
  • a lipid bilayer is positioned in the opening (13).
  • the cavities are filled with liquid, wherein the lipid bilayer separates two differently composed solutions.
  • FIG. 7 shows schematically how in the double or triple chamber systems according to the invention with lipid bilayer, the exchange of liquid in certain compartments can be done.
  • the left side shows the system before replacing the liquid.
  • the points of removal and addition are marked by bars with arrows.
  • On the right side the system is shown after partial replacement of the liquid, wherein the newly added liquid is shown in a lighter shade.
  • FIG. 8 shows how the double or triple measuring chamber systems according to the invention can be arranged to arrays and microtiter plates.
  • Figure 8a is a double measuring chamber unit characterized by framing, which is connected to analog arranged further double measuring chambers on a common floor.
  • FIG. 8b a triple measuring chamber characterized by a frame.
  • the disclosed single measuring chamber as exemplified in FIG. 5 shown comprises a first cavity (11) with lateral walls (4) and an opening upwards. Positioned below the first cavity (11) is a second cavity (12), the first being separated from the second cavity by a hydrophobic partition wall (3).
  • the hydrophobic partition wall (3) has a measuring opening (13) with a diameter of 0.1 to 100 ⁇ m.
  • measuring aperture is here meant an aperture or a hole into which a lipid bilayer can be introduced for the subsequent measurement of optical or electrical properties in the aperture or its surroundings.
  • the openings (14, 17), which are not referred to as “measuring orifice”, do not serve to receive a lipid bilayer, but allow fluid exchange and flow of current between the first and second cavities. They therefore preferably have larger diameter than the measuring openings.
  • each of the first and second cavities (11, 12) an electrode of wire is contained.
  • the measuring chamber is designed in such a way that in the Measuring opening (13) according to the methods described above, a lipid bilayer can be positioned, which separates the first from the second cavity. Via the two electrodes, a voltage can be applied through the lipid bilayer.
  • suitable liquids such as electrolyte solutions and physiological buffer systems or cells to be examined can be added.
  • a double measuring chamber comprises, as in the FIGS. 5B and 6A shown by way of example, two juxtaposed measuring chambers, each having a first cavity (11, 15) with lateral walls (4) and openings upwards. Below the two first cavities (11, 15) a continuous second cavity (12) is positioned. A first cavity (15) is connected to the second cavity (12) through an opening (14). The other first cavity (11) is separated from the second cavity (12) by a hydrophobic partition wall (3).
  • the partition wall contains (3) a measuring opening (13) which has a diameter of 0.1 to 100 ⁇ m, so that the first cavity (11) is connected to the second cavity (12).
  • one electrode (21, 22) is contained in each of the first cavities (11) and (15).
  • the second cavity (12) can be designed like a channel and can be easily filled and rinsed.
  • the arrangement of the double measuring chamber allows pressure equalization by changing the amounts of liquid in the cavities. In this way it is avoided that the lipid bilayer is damaged or breaks up.
  • the possibilities of adding and removing liquid are shown schematically in the FIG. 7A shown.
  • a triple measuring chamber according to the invention is exemplary in the FIGS. 5C and 6B shown. It consists of a double measuring chamber with a further first measuring chamber with a first cavity (16), wherein among the three first cavities (11, 15, 16) is a continuous second cavity (12) is present, and the first cavity (16) is connected to the second cavity (12) through an opening (17).
  • the lipid bilayer is positioned in the measurement port (13) which separates the first cavity (11) from the second cavity (12).
  • This arrangement has the advantage that after formation of the lipid bilayer and thus after separation of the cavity (11), a continuous fluid system via the cavities 12, 15 and 16 is given, via which a pressure equalization takes place.
  • the liquid in the second cavity (12) can be exchanged (rinsed) in the presence of the lipid bilayer. This allows, for example, the continuous or stepwise change of the ion concentration, the rinsing and cleaning of the cavity (12) or the addition or removal of additives whose effect on the lipid bilayer or a contacting cell is investigated.
  • FIG. 7B shows schematically how in a triple-chamber system according to the invention the liquid in the middle first cavity can be exchanged in the presence of the lipid bilayer. In the middle first cavity (cis), the addition and removal takes place in the same way, while in the two interconnected first cavities, the addition and removal can take place in different cavities and the pressure is equalized via the lower cavity.
  • the pressure equalization also prevents the lipid bilayers from being changed or even damaged due to the osmotic gradient and thus falsifying the measurement result. Quite generally, it is preferred in the device according to the invention that a pressure equalization takes place, since the lower cavity (12) has at least one opening which is not closed by a bilayer.
  • the measuring chambers can be arranged to form microtiter plates.
  • the microtiter plates of the present invention have at least two measuring chambers according to the invention.
  • the measuring chambers are arranged in an identical manner in two spatial directions ( FIG. 8 ).
  • the chambers contain an optical access from below, for example by means of a quartz glass pane, and a pipetting access from above.
  • the preparation of the chambers can be done in a cost-effective sandwich construction. This ensures a one-way approach, as needed for pharmacological experiments.
  • With the devices according to the invention it is possible, while maintaining the SBS-compliant microtiter plates, to simultaneously and individually perform both electrical and optical measurements in high-throughput on horizontal membranes. By parallelized measuring chambers in two spatial directions, a large number can be accommodated in a small space.
  • the measuring chambers shown in the figures are shown only schematically, in particular in the figures, the proportions of the components do not necessarily correspond to the real conditions.
  • the bottom (1) has a vertical diameter of 5-200, in particular 10-100 ⁇ m
  • the layer 2 has a diameter of 5-200 ⁇ m, in particular 10-100 ⁇ m
  • the dividing wall (3) has a diameter of 1-100 ⁇ m, in particular 5 to 25 microns.
  • the height of the lateral walls (4) is preferably 0.5 to 10, in particular 1 to 5 mm and the horizontal diameter of the first cavity 0.5 to 10, in particular 1 to 5 mm.
  • the first wells absorb preferably 1 ⁇ l to 1 ml of liquid.
  • the second cavity (12) preferably has the same vertical diameter as the layer 2.
  • the width of the second cavity may vary, preferably between 0.1 and 500 microns.
  • the measuring chamber has the disadvantages that the silver layer impairs optical measurements and that the existing agarose layer is relatively expensive to produce and also degenerates in a period of hours due to loss of water.
  • the device is therefore not suitable in particular as the basis for an automated method.
  • Microtiter plates with volumes on the microliter scale are required in the current high throughput (HTS) and ultra high throughput (UHTS) series of drug discovery in drug discovery.
  • HTS high throughput
  • UHTS ultra high throughput
  • Known electrical and coupled electro-optical measuring methods lack HTS- and UHTS-compatible measuring chambers according to the SBS standard (Society for Biomolecular Screening).
  • the double and triple measuring chambers and microtiter plates of the invention are particularly suitable for detecting properties of cells according to the methods of the invention and for producing the measuring devices according to the invention, in which at least one cell contacts the lipid bilayer.
  • the embodiments of the cell measuring devices according to the invention are therefore in particular features of the double or triple measuring chambers of the invention.
  • the measuring chambers and microtiter plates according to the invention have numerous advantages over the known devices for the examination of lipid bilayers.
  • the measuring chambers according to the invention make it possible to optically and / or electrically track processes between two aqueous phases which are separated from one another by a horizontal lipid double layer in a method according to the invention. It will be the use miniaturized measuring chambers and the use of a sandwich construction, in which a large number of measuring chambers are produced with a small number of components, allows.
  • An optical access is ensured by the bottom of the device.
  • the pipette and electrode access is ensured from above, so that the measuring method can be carried out in a simple manner and also automatically.
  • the microtiter plate according to the invention offers the possibility of performing simultaneous optical or electrical measurements in two aqueous compartments separated from one another by a horizontal lipid bilayer membrane.
  • the electrical and optical properties in the different cavities can be detected individually.
  • the microtiter plates according to the invention offer the possibility of measuring properties of a membrane or membrane processes electrically and / or optically, without losing the character of a UHTS measuring chamber plate.
  • the structure preferably corresponds to a sandwich construction of various suitable materials such as Teflon and glass. Such a layer structure is for example in the FIG. 1 shown (layers 1, 2, 3 and 4).
  • FIG. 5 A preferred embodiment of a double and triple measuring chamber according to the invention is the FIG. 5 refer to.
  • one (or more) microstructured film is inserted between the upper part of the measuring chambers and the glass bottom.
  • a double chamber triple chamber, etc., microtiter plate
  • a hole with a small diameter (0.1-100 ⁇ m) and a hole with a preferably larger diameter and a cavity (channel) connecting both holes horizontally are introduced into the foil.
  • This cavity can be integrated, for example, in the film or in the quartz glass.
  • the separating membrane is mounted horizontally within the depth of focus above the glass layer.
  • the single measuring chamber structure according to the invention with two superimposed cavities does not reduce the number of chambers when arranged to form a microtiter plate. For reasons of stability, it should be used primarily at symmetrical or similar ion concentrations in both cavities, since pressure compensation between the cavities is possible only by deforming the membrane.
  • FIG. 2 shows voltage leads ("Voltage Clamp”) on a horizontal bilayer (right picture) after applying voltage pulses ("voltage gate", picture top left).
  • I V cmd / R bilayer
  • At least one SF-9 cell is applied to this bilayer ( FIG. 3 ).
  • Settling and manipulation of single or multiple cells is accomplished by micropipettes and micromanipulators. It is then after a successful capacitive and ohmic coupling in FIG. 3 shown current response with significant (> 40 times compared with FIG. 2 ) larger mean currents when applying voltage gates measured.
  • SF-9 cells are a cell line derived from insects ( Spodoptera frugiperda ) that are used for the heterologous expression of proteins.
  • FIG. 4 The quantitative verification of the coupling of cells on a horizontal bilayer using a Na + -dependent glutamate transporter (EAAC1) heterologously expressed in Hek cells is shown in FIG. 4 shown.
  • FIG. 4a shows a horizontal bilayer with an EAAC1 Hek 293 cell and the current response of this measurement setup.
  • the glutamate transporter is strictly Na + -dependent in its activity (Grewer et al., 2000). After the Na + ions have been removed by perfusion in both aqueous compartments, the average current is based on the value of bilayers without a coupled cell ( Figure 4c ). at renewed addition of Na + ions, the average currents of the output measuring arrangement are achieved ( Figure 4c ).

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Claims (12)

  1. Procédé pour détecter des propriétés de cellules au moyen d'un dispositif de mesure comprenant deux chambres séparées par une paroi de séparation hydrophobe, la paroi de séparation présentant une ouverture horizontale, dans lequel
    - une bicouche lipidique composée de lipides synthétiques et/ou naturels purifiés est positionnée dans l'ouverture, laquelle bicouche ferme l'ouverture,
    - une solution tampon physiologique est introduite dans les deux chambres,
    - au moins une cellule est mise en contact avec un côté de la bicouche lipidique, pour former un couplage ohmique ou capacitif et
    - des signaux fluorescents sont mesurés au moyen d'un fond optiquement transparent du dispositif de mesure et des électrodes sont disposées dans les chambres et des propriétés électriques sont mesurées.
  2. Procédé selon la revendication 1, caractérisé en ce que le procédé est réalisé de manière automatique.
  3. Chambre de mesure double, comprenant deux chambres de mesure disposées côte à côte, qui contiennent chacune une première cavité (11, 15) avec des parois latérales (4), caractérisée en ce que
    - une deuxième cavité perméable (12) comprenant un fond (1) est positionnée sous les deux premières cavités (11, 15),
    - la première cavité (15) est reliée à la deuxième cavité (12) par une ouverture (14),
    - la première cavité (11) est séparée de la deuxième cavité (12) par une paroi de séparation hydrophobe (3),
    - la paroi de séparation (3) contient une ouverture de mesure horizontale (13) qui présente un diamètre de 0,1 à 100 µm, de sorte que la première cavité (11) est reliée à la deuxième cavité (12) et qu'une bicouche lipidique composée de lipides synthétiques et/ou naturels purifiés est tendue à travers l'ouverture de mesure (13),
    - une électrode (21, 22) est contenue dans chacune des premières cavités (11) et (15) et
    - le fond (1) est composé d'un matériau optiquement transparent.
  4. Chambre de mesure triple composée d'une chambre de mesure double selon la revendication 3 et en plus d'une troisième chambre de mesure comprenant une première cavité (16), dans laquelle
    - une deuxième cavité perméable (12) est positionnée sous les premières cavités (11, 15, 16), et
    - la première cavité (16) est reliée à la deuxième cavité par une ouverture (17).
  5. Chambre de mesure double ou triple selon l'une quelconque des revendications 3 ou 4, caractérisée en ce que l'ouverture (14) ou (17) présente un diamètre de 10 µm à 5 mm.
  6. Chambre de mesure double ou triple selon l'une quelconque des revendications 3 à 5, caractérisée en ce que la paroi de séparation hydrophobe (3) est un film polymère.
  7. Chambre de mesure double ou triple selon l'une quelconque des revendications 3 à 5, caractérisée en ce que la paroi de séparation (3) est composée de Téflon, de polycarbonate, de PMMA, de PDMS ou de Topas.
  8. Chambre de mesure double ou triple selon l'une quelconque des revendications 3 à 7, caractérisée en ce qu'un circuit est réalisé à travers le dispositif de mesure.
  9. Réseau pour détecter automatiquement des propriétés de cellules ou de vésicules, composé d'au moins deux dispositifs de mesure selon l'une quelconque des revendications 3 à 8.
  10. Plaque de microtitrage, composée d'au moins deux chambres de mesure double ou triple selon l'une quelconque des revendications 3 à 8, dans laquelle les deuxièmes cavités (12) sont reliées de manière perméable.
  11. Procédé pour détecter des propriétés de cellules, de vésicules ou de bicouches lipidiques au moyen d'une chambre de mesure double ou triple selon l'une quelconque des revendications 3 à 8 ou d'une plaque de microtitrage selon la revendication 10.
  12. Procédé selon la revendication 11, caractérisé en ce que le procédé est réalisé de manière automatique.
EP05801676A 2004-11-05 2005-11-04 Dispositif et procede pour mesurer les proprietes de cellules Active EP1807696B1 (fr)

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EP05801676A EP1807696B1 (fr) 2004-11-05 2005-11-04 Dispositif et procede pour mesurer les proprietes de cellules

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EP04026252 2004-11-05
EP04030135 2004-12-20
EP05801676A EP1807696B1 (fr) 2004-11-05 2005-11-04 Dispositif et procede pour mesurer les proprietes de cellules
PCT/EP2005/055752 WO2006048447A1 (fr) 2004-11-05 2005-11-04 Dispositif et procede pour mesurer les proprietes de cellules

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EP1807696B1 true EP1807696B1 (fr) 2013-01-09

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EP (1) EP1807696B1 (fr)
JP (1) JP2008518594A (fr)
CA (1) CA2585763A1 (fr)
WO (1) WO2006048447A1 (fr)

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DE102007030378A1 (de) * 2007-06-29 2009-01-02 Spatial View Gmbh Dresden System zur Bestimmung der Lage eines Kamerasystems
JP5359642B2 (ja) * 2009-07-22 2013-12-04 東京エレクトロン株式会社 成膜方法
DE102011120394B4 (de) 2011-12-06 2015-06-25 Universitätsklinikum Freiburg Verfahren und Mikrostrukturvorrichtung zur elektrischen Kontaktierung biologischer Zellen

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US7244349B2 (en) * 1997-12-17 2007-07-17 Molecular Devices Corporation Multiaperture sample positioning and analysis system
EP1040349B2 (fr) * 1997-12-17 2012-12-19 Ecole Polytechnique Federale De Lausanne (Epfl) Positionnement et caracterisation electrophysiologique de cellules individuelles et de systemes membranaires reconstitues sur des supports microstructures
US6682649B1 (en) * 1999-10-01 2004-01-27 Sophion Bioscience A/S Substrate and a method for determining and/or monitoring electrophysiological properties of ion channels
JP2003511679A (ja) * 1999-10-08 2003-03-25 ザ・ボード・オブ・トラスティーズ・オブ・ザ・レランド・スタンフォード・ジュニア・ユニバーシティ 脂質二重層アレイおよびその製造および使用方法
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Publication number Publication date
EP1807696A1 (fr) 2007-07-18
JP2008518594A (ja) 2008-06-05
US20090023600A1 (en) 2009-01-22
CA2585763A1 (fr) 2006-05-11
US20120214708A1 (en) 2012-08-23
WO2006048447A1 (fr) 2006-05-11

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